Essential oils inhibit the bovine respiratory pathogens Mannheimia haemolytica, Pasteurella multocida and Histophilus somni and have limited effects on commensal bacteria and turbinate cells in vitro.

AIMS: The objective of this study was to determine antimicrobial activities of essential oils (EOs) against bovine respiratory disease (BRD) pathogens and nasopharyngeal commensal bacteria, as well as cytotoxicity in bovine turbinate (BT) cells in vitro. METHODS AND RESULTS: The chemical composition of 16 EOs was determined using gas chromatography-mass spectrometry. All EOs were first evaluated for growth inhibition of a single BRD pathogen Mannheimia haemolytica serotype 1 strain (L024A). The most inhibitory EOs (n = 6) were then tested for antimicrobial activity against multidrug-resistant strains of M. haemolytica (serotypes 1, 2 and 6); the BRD pathogens Pasteurella multocida and Histophilus somni, as well as commensal bacteria that were isolated from the nasopharynx of feedlot cattle. The cytotoxicity of 10 EOs was also evaluated using a BT cell line. The EOs ajowan, thyme and fennel most effectively inhibited all BRD pathogens tested including multidrug-resistant strains with minimum inhibitory concentrations (MIC) of ≤0·025% (volume/volume, v/v). For these EOs, the MIC was 2-32 fold greater against commensal bacteria, compared to BRD-associated pathogens. No cytotoxic effects of EOs against BT cells were observed within the tested range of concentrations (0·0125-0·4%, v/v). CONCLUSIONS: The EOs ajowan, thyme and fennel inhibited M. haemolytica, P. multocida and H. somni at a concentration of 0·025% and had minimal antimicrobial activity against nasopharyngeal commensal bacteria and cytotoxicity against BT cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that EOs may have potential for intra-nasal administration to mitigate bovine respiratory pathogens in feedlot cattle.

A vapour phase assay for evaluating the antimicrobial activities of essential oils against bovine respiratory bacterial pathogens.

The objectives of this study were to develop a new assay for the evaluation of the antimicrobial activities of essential oils (EOs) in vapour phase and to demonstrate the antimicrobial activities of commercial EOs against BRPs. To achieve the first objective, a microtube cap containing 100 µl of EO was embedded in an agar plate. An agar plug (diameter 13 mm) inoculated with a bacterial suspension containing108  CFU per ml was then placed over the cap and incubated at 37°C for 24 h. Subsequently, bacteria were recovered from the agar plug by immersion in 5 ml of broth for 10 min, followed by vortexing for 30 s, and the broths were then plated for enumeration. To demonstrate the usefulness of the assay, nine commercial EOs derived from the following specific plants: ajowan, carrot seed, cinnamon leaf, citronella, fennel, ginger grass, lavender, rosemary and thyme were first evaluated for their vapour phase antimicrobial activities against Mannheimia haemolytica serotype 1. Selected EOs were further tested against Pasteurella multocida and Histophilus somni. The EOs of ajowan, thyme and cinnamon leaf completely or partially inhibited BRPs growth. This new assay provided reproducible results on the vapour phase antimicrobial activities of EOs against BRPs. These results support further study of EOs as a potential mitigation strategy against BRPs. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we present a new vapour phase assay for evaluating the antimicrobial activities of essential oils (EO) against bovine respiratory pathogens (BRPs). Using this assay, we identified EOs, such as ajowan, thyme and cinnamon leaf, that can effectively inhibit growth of the BRPs Mannheimia haemolytica serotype 1, Pasteurella multocida and Histophilus somni. This is the first study to demonstrate the vapour phase antimicrobial activity of EOs against BRPs.

Probiotic bacteria inhibit the bovine respiratory pathogen Mannheimia haemolytica serotype 1 in vitro.

This study evaluated the potential of probiotic bacteria to inhibit growth and cell adhesion of the bovine respiratory pathogen Mannheimia haemoltyica serotype 1. The inhibitory effects of nine probiotic strains (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Streptococcus thermophilus and two Paenibacillus polymyxa strains) against M. haemolytica were evaluated using a spot-on-lawn method. Probiotic strains were then tested for their adherence to bovine bronchial epithelial (BBE) cells and the ability to displace and compete against M. haemolytica on BBE. Except for S. thermophilus, all probiotic strains inhibited the growth of M. haemolytica, with zones of inhibition ranging between 12 and 19 mm. Lactobacillus strains and Lactococcus lactis displayed greater (P < 0·05) BBE adhesion compared with M. heamolytica (8·3%) and other probiotics (<2·2%). Strains of P. polymyxa and L. acidophilus caused the greatest reduction in M. haemolytica adherence, through both displacement and competition, compared with other probiotics. The results of this study suggest that probiotics may have the potential to colonize the bovine respiratory tract, and exert antagonistic effects against M. haemolytica serotype 1. SIGNIFICANCE AND IMPACT OF THE STUDY: A common method to control bovine respiratory disease (BRD) in feedlots is through mass medication with antibiotics upon cattle entry (i.e. metaphylaxis). Increasingly, antimicrobial resistance in BRD bacterial pathogens has been observed in feedlots, which may have important implications for cattle health. In this study, probiotic strains were shown to adhere to bovine respiratory cells and inhibit the BRD pathogen M. haemolytica serotype 1 through competition and displacement. Probiotics may therefore offer a mitigation strategy to reduce BRD bacterial pathogens, in place of metaphylactic antimicrobials.

A multiplex PCR assay for the detection and quantification of Sclerotinia sclerotiorum and Botrytis cinerea.

UNLABELLED: Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A primer set (SsIGS_5) for S. sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S. sclerotiorum strains and no other species. For qPCR, the SsIGS_5 primer and probe set exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2)  = 0·99). SsIGS_5 was then multiplexed with a previously published primer/probe set for B. cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S. sclerotiorum and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa fields. SIGNIFICANCE AND IMPACT OF THE STUDY: A primer and probe set for the quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The probe-based nature of this assay signifies an improvement over previous assays for this species by allowing multiplex reactions while maintaining high sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid analysis of environmental samples. In crops susceptible to both pathogens, this multiplex assay can be used to quickly quantify the presence of each pathogen.

Characterization of tetracycline resistance genes in Escherichia coli isolated from feedlot cattle administered therapeutic or subtherapeutic levels of tetracycline.

The effect of administering feedlot cattle subtherapeutic levels of chlortetracycline (CT) or CT and therapeutic levels of oxytetracycline (CT-OX) on resistance genotypes in Escherichia coli was investigated. Detection of genes tet(A), tet(B), and tet(C) encoded by tetracycline-resistant isolates (CT, N = 77; CT-OX, N = 99) was performed by multiplex polymerase chain reaction (PCR). Prevalence of tet(A) was similar in isolates across treatment regimes; however, prevalence of tet(B) was lower (18% versus 34%; P < 0.05) and tet(C) was higher (46% versus 28%; P < 0.05) in CT isolates compared with CT-OX isolates. To further characterize selection of resistance genotypes in E. coli, a group of intermediately tetracycline-resistant E. coli (N = 48) was analyzed. The tet(C) gene was present in 92% of these isolates. Copies of tet(C) transcripts, analyzed by real-time PCR, indicated that upregulation did not occur in tetracycline-resistant isolates when compared with intermediately resistant isolates. The minimum inhibitory concentrations of tetracycline, chlortetracycline, and oxytetracycline were also tested on isolates with different resistance genes. The minimum inhibitory concentration was dependent on the tetracycline analogue and the nature of encoded resistance. These data indicate that tetracycline analogues should not be used interchangeably to evaluate resistance and that prevalence of resistance genes in E. coli can vary according to the tetracycline analogue administered to cattle.

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity.

AIMS: This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle. METHODS AND RESULTS: Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae- or Siphoviridae-like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·0001). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70-79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2-like phage φMhaA1-PHL101. CONCLUSIONS: Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.

A fibrolytic enzyme additive for lactating Holstein cow diets: ruminal fermentation, rumen microbial populations, and enteric methane emissions.

The objective was to determine if supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive (Econase RDE; AB Vista, Marlborough, Wiltshire, UK) altered fermentation, pH, and microbial populations in the rumen or enteric methane (CH(4)) emissions. In a companion study, this enzyme additive improved efficiency of fat-corrected milk production in a dose-dependent manner by up to 11% for early lactation dairy cows. Nine ruminally cannulated, lactating Holstein cows were used in a replicated 3 × 3 Latin square design with 21-d periods. Dietary treatments were 0 (control), 0.5 (low), and 1.0 (high) mL of enzyme/kg of total mixed ration dry matter. Rumen contents were collected on 2 d (d 15 and 19), ruminal pH was measured continuously for 6 d (d 13 to 18) by using an indwelling system, and enteric CH(4) production was measured for 3 d (d 16 to 18) using the sulfur hexafluoride tracer gas technique. The enzyme additive did not alter volatile fatty acids, NH(3), pH, or population densities of total protozoa, bacteria, and methanogens in ruminal fluid. However, population densities of certain bacteria, calculated as copy number of species-specific 16S-rRNA, were affected by enzyme treatment. Population density of Ruminobacter amylophilus was increased and that of Fibrobacter succinogenes tended to be increased by the high enzyme treatment. Selenomonas ruminantium tended to increase linearly with increasing levels of enzyme in the diet, although its population density was only numerically increased by the high enzyme treatment. Streptococcus bovis, however, tended to be decreased by the low enzyme treatment. Increasing the level of enzyme supplement in the diet also linearly increased enteric CH(4) production, even when adjusted for feed intake or milk production (19.3, 20.8, and 21.7 g of CH(4)/kg of dry matter intake or 12.9, 13.6, and 15.1g of CH(4)/kg of milk for the control, low, and high enzyme treatments, respectively). This shift in ruminal bacterial communities and higher CH(4) emissions could imply increased ruminal digestion of feed, which needs to be substantiated in longer term studies.

Prevalence and diversity of class 1 integrons and resistance genes in antimicrobial-resistant Escherichia coli originating from beef cattle administered subtherapeutic antimicrobials.

AIMS: To characterize class 1 integrons and resistance genes in tetracycline-resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control). METHODS AND RESULTS: Tetracycline-resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher (P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 (r = 0·44), aadA1 (r = 0·61), cat (r = 0·58) and intI1(r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays. CONCLUSIONS: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline-resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline-resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline-resistant E. coli isolated from feedlot cattle.

Genetic characterization and antimicrobial susceptibility of Mannheimia haemolytica isolated from the nasopharynx of feedlot cattle.

A surveillance study was undertaken to examine the population dynamics and antimicrobial resistance of Mannheimia haemolytica isolated from feedlot cattle. A total of 416 isolates were collected from the nasopharynx either upon entry or exit from two feedlots in southern Alberta, Canada. Isolates were serotyped, characterized by pulsed-field gel electrophoresis and tested for susceptibility to ten antimicrobial agents via disk diffusion. Resistant isolates were screened by PCR for select antimicrobial-resistance gene determinants. Isolates were highly diverse, with 335 unique pulsed-field profiles identified among 147 strongly related clusters (similarity ≥ 85%). Clonal spread of isolates throughout the feedlots was limited and no clear association was found between genetic relatedness of M. haemolytica and sampling event (entry or exit). Pulsed-field profiles sharing a common serotype and resistance phenotype tended to cluster together. The majority of isolates were identified as serotype 2 (74.5%) although both serotype 1 (11.9%) and 6 (12.7%) were detected. Only 9.54% of isolates exhibited antimicrobial resistance. Resistance to oxytetracycline was most prevalent (n=16), followed by ampicillin (n=10), and amoxicillin/clavulanic acid (n=7). Multi-drug resistance was observed in five isolates. The tetH gene was detected in all but two oxytetracycline resistant isolates. Other detectable resistance determinates included ermX and bla(ROB-1). In the two feedlots examined, M. haemolytica exhibited considerable genetic diversity and limited resistance to common veterinary antibiotics. Garnering further information on the linkage between genotype and phenotype should contribute toward a better understanding of the pathogenesis and dissemination of M. haemolytica in feedlots.

Survival of Escherichia coli O157:H7 in ruminal or fecal contents incubated with corn or wheat dried distillers' grains with solubles.

Dried distillers' grain with solubles (DDGS) is a by-product of ethanol production, and its use as cattle feed has increased as a result of the expansion of the fuel ethanol industry. However, the inclusion of corn DDGS into feedlot diets may increase the shedding of Escherichia coli O157:H7. This study investigated whether corn or wheat DDGS at 2 concentrations (20% or 40% vs. 100% barley grain) affected the survival of E. coli O157:H7 in incubations of ruminal digesta and feces. Neither the type nor the level of DDGS had any effect on fermentation or the survival of E. coli O157:H7 in ruminal digesta. However, there was a time by DDGS interaction (p < 0.05), where the numbers of E. coli O157:H7 in feces did not differ after 4 or 12 h of incubation but were greater after 24 h in both 40% wheat and 40% corn DDGS as compared with other treatments. Additionally, after 24 h, the numbers of E. coli O157:H7 were greater in fecal incubations with corn DDGS than with wheat DDGS (p < 0.05). The differences in the numbers of E. coli O157:H7 were not attributable to changes in pH or in concentrations of volatile fatty acids in the media. These results suggest that the inclusion of high levels of corn or wheat DDGS in feedlot diets of cattle may encourage the survival of E. coli O157:H7 in feces.

Comparison of repetitive PCR and pulsed-field gel electrophoresis for the genotyping of Mannheimia haemolytica.

Mannheimia haemolytica is an opportunistic pathogen that can cause fibrinonecrotic pneumonia in cattle and is the main bacterial agent implicated in bovine respiratory disease-complex (BRD). Despite its economic importance to the cattle industry, few studies have characterized the genetic nature of M. haemolytica and none have genotyped isolates from feedlots. Identifying and monitoring genetic variants of M. haemolytica is important to understanding the etiology of BRD in cattle. We investigated the capacity of three genotyping techniques (BOX-PCR, (GTG)(5)-PCR and PFGE analysis of SalI-restricted DNA) to discriminate among 24 reference strains from the family Pasteurellaceae and 40 M. haemolytica isolates collected from feedlot cattle. From cluster analysis of the M. haemolytica isolates, PFGE was revealed as most discriminating, followed by BOX-PCR and then (GTG)(5)-PCR (Simpson's diversity index >0.98, 0.82, and 0.72, respectively). Of these methods, PFGE also had the greatest mean repeatability (0.96). The PFGE and BOX-PCR assays grouped all M. haemolytica in a single cluster but only BOX-PCR and (GTG)(5)-PCR grouped the Mannheimia glucosida and Mannheimia ruminalis strains together. Refinement of genotyping procedures for M. haemolytica could offer new insight into the etiology of this pathogen in BRD.

Farm-to-fork characterization of Escherichia coli associated with feedlot cattle with a known history of antimicrobial use.

This study investigated antimicrobial-resistant (AR) Escherichia coli isolated from "farm-to-fork" production of cattle fed diets containing the antimicrobial growth promoter (AGP) chlortetracycline plus sulfamethazine (44ppm each, AS700) or no AGP (control). For each treatment, samples included: feces just prior to euthanization; hides after euthanization; intestinal digesta from the lower digestive tract; carcasses immediately after evisceration and after 24h in the chiller; and ground beef stored at 5 degrees C for 1 and 8days. Samples were also collected from the abattoir environment and from air during hide removal. Total, ampicillin (Amp(r))-, and tetracycline (Tet(r))-resistant E. coli were isolated on MacConkey agar or MacConkey agar containing ampicillin or tetracycline, respectively. Amp(r) and Tet(r)E. coli were isolated from the feces and hides of all cattle. Compared to the control, the prevalence of Amp(r) (26.5% vs. 7.9%) and Tet(r) (50.9% vs. 12.6%) E. coli was greater in feces from AS700 treated animals (P<0.05), but was similar between treatments for hide samples (P>0.05). The prevalence of carcass or ground beef contamination with AR E. coli was not different between treatments. Resistant E. coli were isolated from the abattoir environment after processing of both groups of cattle. Susceptibilities to 11 antimicrobials and pulsed-field gel electrophoresis (PFGE) analyses were conducted on 360 Amp(r) and Tet(r)E. coli isolates. Twenty-five antibiogram profiles were detected, with isolates exhibiting resistance to up to 9 antimicrobials. Most (28.2%) Amp(r)E. coli were also resistant to streptomycin and tetracycline, whereas Tet(r)E. coli (53.5%) were mainly resistant to only tetracycline. Thirty one genotypes were detected by PFGE with most isolates from meat and environmental samples having similar genetic profiles to isolates from hides or digesta. These data demonstrate that antimicrobial-resistant E. coli can contaminate meat products during slaughter and enter the food chain regardless of whether or not cattle are administered AGP. The abundance of AR E. coli on the hides of animals is likely a key element for controlling end-product contamination.

Recovery of antimicrobial-resistant Escherichia coli after storage of bovine feces in Cary-Blair medium.

The effect of storing bovine feces in Cary-Blair medium on the recovery of antimicrobial-resistant Escherichia coli was investigated. Feces from cattle at a research feedlot (n = 50) and at a commercial feedlot (n = 46) were processed immediately or after storage in Cary-Blair medium for 8 days at 5 degrees C. Total, ampicillin-resistant, and tetracycline-resistant E. coli were isolated. The number of total E. coli decreased slightly after storage (0.19 log units; p < 0.001), but storage of feces in Cary-Blair medium did not affect recovery of ampicillin- or tetracycline-resistant E. coli.

Longitudinal characterization of resistant Escherichia coli in fecal deposits from cattle fed subtherapeutic levels of antimicrobials.

Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Amp(r)) and tetracycline-resistant (Tet(r)) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Amp(r) and Tet(r) E. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of Tet(r) E. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tet(r) and Amp(r) E. coli. Except for Amp(r) E. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high Tet(r) E. coli numbers were detected in A44 and AS700 deposits [5.9 log(10) CFU (g dry matter)(-1) and 5.4 log(10) CFU (g dry matter)(-1), respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, bla(TEM1), tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.

Effect of subtherapeutic administration of antibiotics on the prevalence of antibiotic-resistant Escherichia coli bacteria in feedlot cattle.

Antibiotic-resistant Escherichia coli in 300 feedlot steers receiving subtherapeutic levels of antibiotics was investigated through the collection of 3,300 fecal samples over a 314-day period. Antibiotics were selected based on the commonality of use in the industry and included chlortetracycline plus sulfamethazine (TET-SUL), chlortetracycline (TET), virginiamycin, monensin, tylosin, or no antibiotic supplementation (control). Steers were initially fed a barley silage-based diet, followed by transition to a barley grain-based diet. Despite not being administered antibiotics prior to arrival at the feedlot, the prevalences of steers shedding TET- and ampicillin (AMP)-resistant E. coli were >40 and <30%, respectively. Inclusion of TET-SUL in the diet increased the prevalence of steers shedding TET- and AMP-resistant E. coli and the percentage of TET- and AMP-resistant E. coli in the total generic E. coli population. Irrespective of treatment, the prevalence of steers shedding TET-resistant E. coli was higher in animals fed grain-based compared to silage-based diets. All steers shed TET-resistant E. coli at least once during the experiment. A total of 7,184 isolates were analyzed for MIC of antibiotics. Across antibiotic treatments, 1,009 (13.9%), 7 (0.1%), and 3,413 (47.1%) E. coli isolates were resistant to AMP, gentamicin, or TET, respectively. In addition, 131 (1.8%) and 143 (2.0%) isolates exhibited potential resistance to extended-spectrum beta-lactamases, as indicated by either ceftazidime or cefpodoxime resistance. No isolates were resistant to ciprofloxacin. The findings of the present study indicated that subtherapeutic administration of tetracycline in combination with sulfamethazine increased the prevalence of tetracycline- and AMP-resistant E. coli in cattle. However, resistance to antibiotics may be related to additional environmental factors such as diet.

Prospective study of biliary strictures to determine the predictors of malignancy.

BACKGROUND: There have been few prospective studies regarding the investigation of biliary strictures, principally because of rapid technological change. The present study was designed to determine the sensitivity of various imaging studies for the detection of biliary strictures. Serum biochemistry and imaging studies were evaluated for their role in distinguishing benign from malignant strictures. METHODS: Thirty-one patients with suspected noncalculus biliary obstruction were enrolled consecutively in the study. A complete biochemical profile, ultrasound, Disida scan and cholangiogram (endoscopic retrograde cholangiopancreatography [ERCP] or percutaneous cholangiogram) were obtained at study entry. Stricture etiology was determined based on cytology, biopsy and/or clinical follow-up at one year. RESULTS: Twenty-nine of 31 patients had biliary strictures, of which 15 were malignant. The mean age of the malignant cohort was 73.9 years versus 53.9 years in the benign cohort (P<0.001). Statistically significant differences between the malignant and benign groups, respectively, were as follows: alanine transaminase 235.2 versus 66.9 U/L (P=0.004), aspartate transaminase 189.8 versus 84.5 U/L (P=0.011), alkaline phosphatase 840.2 versus 361.1 U/L (P=0.002), bilirubin 317.8 versus 22.1 micromol/L (P<0. 001) and bile acids 242.5 versus 73.2 micromol/L (P=0.001). Threshold analysis using receiver operative characteristic (ROC) curves demonstrated that a bilirubin level of 75 micromol/L was most predictive of malignant strictures. Intrahepatic duct dilation was present in 93% of malignant strictures versus 36% of benign strictures (P=0.002). Common hepatic duct dilation was less discriminatory (malignant 13.5 versus benign 9.6 mm; P=0.11). Ultrasound was highly sensitive (93%) in the detection of the primary tumour in the bile duct or pancreas, or in the visualization of nodal or liver metastases. In benign disease, ultrasound failed to detect evidence of intrahepatic or extrahepatic biliary dilation in most cases. Disida scans were not able to distinguish between malignant or benign strictures and could not accurately localize the level of obstruction. The sensitivity of Disida scan for the diagnosis of obstruction was 50%. Cholangiographic characterization of strictures revealed an equal distribution of smooth (eight of 13) and irregular (five of 13) strictures in the malignant group. Ten of 13 benign strictures were characterized as smooth. Malignant strictures were significantly longer than benign ones - 30.3 versus 9.2 mm (P=0.001). Threshold analysis using ROC curves showed that strictures greater than or equal to 14 mm were predictive of malignancy (sensitivity 78%, specificity 75%, log odds ratio 11.23). CONCLUSIONS: A serum bilirubin level of 75 micromol/L or higher, or a stricture length of greater than 14 mm was highly predictive of malignancy in patients with a biliary stricture. Ultrasound was useful in predicting malignant strictures by detecting either intrahepatic duct dilation or by visualizing the tumour (primary or metastases). Strictures with a 'benign' cholangiographic appearance are frequently malignant. Disida scan did not add additional information. ERCP is necessary to diagnose benign strictures, which tend to be less extensive at presentation.

Transfemoral digital subtraction aortography. Are diluted high osmolar contrast media acceptable?

The ionic monomer, sodium diatrizoate at 150 mg I/ml (726 mosmol/kg) and the non-ionic monomer, iopamidol, diluted to the same iodine concentration but at 324.3 mosmol/kg, were randomly allocated to patients undergoing transfemoral intra-arterial digital subtraction angiography for lower limb peripheral vascular disease. The agents produced images of comparable quality and diagnostic efficacy. There were no significant differences between the media regarding sensations of pain and warmth. Minor neurological symptoms (headache and dizziness) occurred 7 times more frequently with the ionic monomer. There was a slight but temporary rise in plasma potassium one hour after injection of the ionic monomer but no evidence of appreciable intravascular haemolysis. The non-ionic monomer caused a slight fall in haemoglobin and haematocrit one hour after injection which is attributed to osmotic haemodilution. It is concluded that a diluted high osmolar contrast agent is an acceptable alternative to a low osmolar agent in transfemoral digital subtraction lower limb aortography.